The tumor suppressor p53 is a critical guardian of somatic cells in the face of cellular stress and genomic challenges and also functions during late embryogenesis, differentiation and senescence. The mechanisms dictating and regulating these p53-mediated responses, and if they occur in human (h)ES cells, are unknown. Our hypothesis is that mechanisms that induce loss of pluripotency also activate p53, which functions by association with specific proteins, binding to target genes and effecting chromatin modification and regulation of target gene expression in hES cells. To test this hypothesis, our studies are guided by the following specific aims: Specific Aim 1: To define the status of p53 in human EScells. Levels of p53 vary considerably between mES and hES cells; therefore, we cannot assume that p53 is regulated or functions in hES cells in the same manner as in mES cells. To explore these unknowns, we will determine expression patterns and protein localization, during stem cell maintenance and after RA-treatment. We will correlate the expression of p53 with markers of pluripotency at the level of expression and morphology, in collaboration with Dr. Austin Cooney (Project 2) and Dr. Thomas Zwaka (Project 4). Specific Aim 2: To determine the consequences of p53-mediated regulation in human ES cells. We will determine the direct gene targets of p53 by global and candidate gene analysis of expression and p53- interactions with chromatin, in collaboration with Dr. Austin Cooney (Project 2). We will further determine if p53 is essential for regulation of specific gene targetsand in maintenance or differentiation of hES cells. Transient knockdown of p53 expression by RNAi-mediated approaches, in collaboration with Dr. Austin Cooney (Project 2) and Dr. Thomas Zwaka (Project 4), and knock-in hES cells, expressing regulated levels of shRNA against p53, will be created in collaboration with hES Genetic Modification core (Core C). Specific aim 3: To determine the mechanisms of p53-mediated regulation in hES cells. We used "knock-in" technology to create mES cell lines that express endogenous p53 protein fused with an epitope (TAP) tag. We will develop TAP-p53 hES cells, where p53 is expressed at very low levels and little is known regarding its functions; underscoring the need for epitope-based methodologies. Knock-in of TAP peptide sequences will done in collaboration with the hES Genetic Modification core (Core C). TAP-purification and mass spectrometry will be used to define a differentiation-specific, p53-proteome of hES cells in collaboration with Dr. Zhou Songyang (Project 1). The consequences of gene-specific association of p53 and interacting partners will be determined by ChIP analysis of binding, histone modifications and determinations of gene expression, prior to and after RA-treatment.